Because of the importance of calcium as a intracellular messenger and regulator, a wide variety of techniques have been developed for measuring intracellular free calcium concentrations [Ca.sup.2+ ].sub.i. The most successful of these techniques use dyes or proteins which change absorption or luminescence upon binding Ca.sup.2+ ions. Currently the most popular of these methods is to monitor the fluorescence of BAPTA-like indicator dye compounds known as quin-2, fura-2, and indo-1. (See references 1-3 and U.S. Pat. No. 4,603,209.) The popularity of compounds such as quin-2, fura-2, and indo-1 stems from the following: (1) the ease with which these compounds can be loaded into cells by hydrolysis of membrane-permeant esters, and (2) the sensitivity and versatility of fluorescence, e.g., a mode of readout adaptable to bulk suspensions, flow cytometry, and microscopic imaging of single cells. Unfortunately, dyes such as quin-2, fura-2, and indo-1 all require excitation at ultraviolet wavelengths, near the cutoff point for transmission through glass. In addition, these wavelengths are potentially injurious to cells and tend to excite auto-fluorescence, for example, from the pyridine nucleotides. In addition, the UV range coincides with the wavelengths needed to photolyse chelators such as nitr-5 and nitr-7, to release their bound Ca.sup.2+. (See references 4-6 and U.S. patent application Ser. No. 049,658, filed May 3, 1987, and U.S. Pat. No. 4,689,432, issued Aug. 25, 1987.) As a result, the existing indicators cannot readily be used to monitor release of "caged" Ca.sup.2+ since the fluorescence excitation will begin to photolyse the buffer. Moreover, the absorbance of nitr-5 or nitr-7 and their photolysis reaction products may cause inner-filtering which can actually perturb the fluorescence excitation.
The problems discussed in the preceding paragraph would be avoided with Ca.sup.2+ indicator dyes whose excitation wavelengths are in the visible or infrared range. There has been a long felt need for these dyes but previous attempts to produce such products resulted in disappointing fluorescent quantum efficiencies or Ca.sup.2+ affinities (See reference 7.)
Fluorescein and rhodamine are fluorophores widely used in biology. Because of their ubiquity as labels in immunofluorescence and fluorescent analog cytochemistry (see reference 8), most fluorescence microscopes and flow cytometers are equipped to handle their wavelengths. As a result, it would be very useful if these highly fluorescent delocalized xanthenes could be combined with proven Ca.sup.2+ specific binding sites of BAPTA or BAPTA-like compounds. Because of their similarity to fluoresceins and rhodamines, such new compounds would be optically compatible with almost any fluorometer or fluorescence microscope or flow cytometer now in use for immunofluorescent detection. Because of the ready availability of such immunofluorescent detection equipment, the new dyes would be welcomed by a number of biological investigators.